Treatment of Human papillomavirus (HPV) infections is a major unmet medical need. Importantly, the licensed HPV vaccines are not able to clear existing infections, and there are now multiple FDA-approved tests to detect high risk HPV DNA, and specifically HPV16. As these HPV DNA tests are widely implemented, large numbers of infected patients are being identified. Currently persistently HPV16+ patients are not treated, but are followed until they develop pre-cancer and then undergo a surgical or ablative intervention with significant costs and side effects. Our overall goal is to develop a therapeutic vaccine to eliminate persistent HPV16 infections. Rather than antibodies, therapeutic HPV vaccines require a potent cytotoxic T cell immune response to control chronic HPV infections and HPV-associated disease. The HPV early proteins E6 and E7 are obligately expressed in all HPV infected cells, absent from normal cells and are 'non-self' antigens. Thus while E6 and E7 are the logical targets for therapeutic HPV vaccination, nevertheless they are weakly immunogenic and require Papivax's novel immunostimulatory technology to generate potent cytotoxic T cell immunity. We have previously utilized a naked DNA vaccine to significantly enhance MHC class I presentation of HPV16 E7 by its fusion to the Mycobacterium tuberculosis heat shock protein 70 (HSP70). This linkage augmented the E7-specific CD8+ T cell immune responses at least 50-fold and led to potent therapeutic effects against HPV16 E7-expressing cells in vaccinated mice. These data led to a phase I clinical trial in HPV16+ patients which demonstrated that immunization with E7HSP70 DNA vaccine induces an E7-specific CD8+ T cell response in patients. TA-HPV is vaccinia virus engineered to express the E6 and E7 genes from HPV16 and HPV18. Cutaneous administration of TA-HPV to nearly a hundred patients with HPV16+ cervical cancer and anogenital neoplasias failed to produce a clear clinical response. Importantly, while priming of mice with the E7HSP70 DNA vaccine followed by cutaneous boosting with recombinant vaccinia expressing HPV E7 enhanced E7-specific CD8+ T cell responses, we recently demonstrated that intralesional boosting with E7- expressing vaccinia was far more effective than either cutaneous boosting or intralesional E7-vaccinia alone. We hypothesize that priming with the E7HSP70 DNA vaccine followed by intralesional injection of TA-HPV vaccinia (which also expresses HPV16 E7) will generate potent therapeutic effects against persistent HPV infection by combining preferential vaccinia replication and cell killing in the lesion with boosting of HPV antigen-specific CD8+ T cell mediated immune responses attracted to the lesion. Thus, we will test whether combining clinical grade HPV DNA vaccine and intra-lesional administration of HPV recombinant vaccinia triggers immune clearance using a new model of persistent HPV16 infection in the mouse vagina. These preclinical studies will drive future Papivax-driven clinical trials of therapeutic vaccines to eliminate persistent HPV infections.